Effective biological control of plant diseases with epiphytic microbes has been documented for numerous phyllosphere- and rhizosphere-inhabiting organisms. Foliar biological control agents include yeast and filamentous fungi (see Hofstein R and A. Chapple, “Commercial development of biofungicides,” Biopesticides: Use and Delivery (Hall F R, Menn J J, eds.), Totowa: Humana Press (1999); and Sutton, J. C. and G. Peng, “Manipulation and vectoring of biocontrol organisms to manage foliage and fruit diseases in cropping systems,” Annual Review of Phytopathology, 31:473-493 (1993)) as well as bacteria; including both gram (−) species such as Erwinia sp. and Pseudomonas sp. (see Andrews, J. H., “Biological control in the phyllosphere,” Annual Review of Phytopathology, 30:603-635 (1992)), and gram (+) organisms such as Bacillus sp. See Kokalis-Burelle, N., P. A. Backman, R Rodriquez-Kabana, and L. D. Ploper, “Potential for biological control of early leafspot of peanut using Bacillus cereus and chitin as foliar amendments,” Biological Control, 2:321-328 (1992). Biological control agents applied to the rhizosphere include Pseudomonads (see Alstrom, S., “Induction of disease resistance in common bean susceptible to halo blight bacterial pathogen after seed bacterisation with rhizosphere pseudomonads,” Journal of Genetic and Applied Microbiology, 37:495-501 (1991); van Peer, R, G. J. Niemann, and B. Schippers, “Induced resistance and phytoalexin accumulation in biological control of fusarium wilt of carnation by Pseudomonasa sp. strain WCS417r,” Phytopathology, 81:728-734 (1991); and van Loon L. C. and C. M. J. Pieterse, “Biological control agents in signaling resistance,” Biological Control of Crop Diseases (Gnanamanickan S S, ed.), New York: Mercel Dekker, Inc, 486 (2002)) as well as Bacillus sp. (see Zhang, S., M. S. Reddy, N. Kokalis-Burelle, L. W. Wells, S. P. Nightengale, and J. W. Kloepper, “Lack of induced systemic resistance in peanut to late leaf spot disease by plant growth-promoting rhizobacteria and chemical elicitors,” Plant Disease, 85(8):879-884 (2001); and Murphy, J. F., G. W. Zehnder, D. J. Schuster, E. J. Sikora, J. E. Polston, and J. W. Kloepper, “Plant growth-promoting rhizobacterial mediated protection in tomato against Tomato mottle virus,” Plant Disease, 84(7):779-784 (2000)) that are classically referred to as plant growth-promoting rhizobacteria. For the most part biological disease control is attributed to direct antagonism against the pathogen via production of antibiotics or hydrolytic enzymes, or through competition for nutrients. See Weller. D. M., “Biological control of soil-borne plant pathogens in the rhizosphere with bacteria.” Annual Review of Phytopathology. 26:379-407 (1988). However, plant growth-promoting rhizobacteria and rhizosphere inhabiting fungi have been shown to stimulate the induction of systemic resistance responses within the plant. See van Peer. R. G. J. Niemann. and B. Schippers, “Induced resistance and phytoalexin accumulation in biological control of fusarium wilt of carnation by Pseudomonasa sp. strain WCS417r.” Phytopathology, 81:728-734. (1991); Wei. G. J. W. Kloepper. and S. Tuzun, “Induction of systemic resistance of cucumber to Colletotrichum orbiculare by select strains of plant growth-promoting rhizobacteria,” Phytopathology, 81:1508-1512 (1991); van Loon, L. C. and C. M. J. Pieterse, “Biological control agents in signaling resistance.” Biological Control of Crop Diseases (Gnanamanickan. S. S., ed.). New York: Mercel Dekker, Inc. 486 (2002). All publications mentioned above are incorporated herein by reference in their entireties for all purposes.
Systemic induced resistance (SIR) has been described in many plant systems, most notably tobacco, bean, tomato, cucumber, and Arabidopsis thaliana. See Ross, A F., “Localized acquired resistance to plant virus infection in hypersensitive hosts,” Virology, 14:329-339 (1961); Kuc, J., “Induced immunity to plant disease,” BioScience, 32:854-860 (1982); Ryals, J. A., U. H. Neuenschwander, M. G. Willits, A. Molina, H. Y. Steiner, and M. D. Hunt, “Systemic acquired resistance,” The Plant Cell. 8:1809-1819 (1996); and van Loon, L. C. and C. M. J. Pieterse, “Biological control agents in signaling resistance.” Biological Control of Crop Diseases (Gnanamanickan. S. S., ed.). New York: Mercel Dekker, Inc. 486 (2002). The broad-spectrum resistance makes an otherwise susceptible plant resistant to a wide array of subsequent pathogen attacks. See Kuc, J. “Induced immunity to plant disease,” BioScience, 32:854-860 (1982); and Hutcheson, S. W., “Current concepts of “active defense in plants.” Annual Review of Phytopathology, 36:59-90 (1998). Elicitation of systemic disease resistance in plants has thus far been achieved through treatment by three types of stimuli: necrotizing pathogens (see Pieterse, C. M. J., S. C. M. van Wees. E. Hoffland, J. A. van Pelt, and L. C. van Loon, “Systemic resistance in Arabidopsis induced by biocontrol bacteria is independent of salicylic acid accumulation and pathogenesis-related gene expression,” The Plant Cell, 8:1225-1237 (1996); Ross, AF., “Localized acquired resistance to plant virus infection in hypersensitive hosts,” Virology, 14:329-339 (1961); Ross. AF., “Systemic acquired resistance induced by localized virus infection in plants,” Virology. 14:340-358 (1961); and Kuc, J., “Induced immunity to plant disease.” BioScience. 32:854-860 (1982)), secondary signal molecules (Le. salicylic acid, SA) (see White, R. F., “Acetylsalicylic acid (aspirin) induces resistance to tobacco mosaic virus in tobacco,” Virology. 99:410-412 (1979)) and their functional analogs (e.g. 2,6 dichloroisonicotinic acid, INA (see Metraux, J. P., P. Ahl-Goy, T. Staub, J. Speich, A Steinemann, J. Ryals, and E. Ward, “Induced resistance in cucumber in response to 2,6-dichloroisonicotinic acid and pathogens,” Advances in Molecular Genetics of Plant-Microbe Interactions, Vol. 1. (H. Hennecke, D. P. S. Verma, eds.), Dordrecht: Kluwer Academic Publishers, 432-439 (1991)) and acibenzolar-5-methyl. ASM (see Tally, A, M. Oostendorp, K. Lawton, T. Staub, and B. Bassi, “Commercial development of elicitors of induced resistance to pathogens,” Induced Plant Defenses Against Pathogens and Herbivores (AA Agrawal, S. Tuzun, and E. Bent, eds.) St. Paul: APS Press, 299-318 (1999)), and plant growth-promoting rhizobacteria introduction into the rhizosphere. See Alstrom, S., “Induction of disease resistance in common bean susceptible to halo blight bacterial pathogen after seed bacterisation with rhizosphere pseudomonads,” Journal of Genetic and Applied Microbiology, 37:495-501 (1991); van Loon, L. C. and C. M. J. Pieterse, “Biological control agents in signaling resistance,” Biological Control of Crop Diseases (Gnanamanickan, S. S., ed.), New York: Mercel Dekker, Inc, 486 (2002); Wei, G., J. W. Kloepper, and S. Tuzun, “Induction of systemic resistance of cucumber to Colletotrichuni orbiculare by select strains of plant growth-promoting rhizobacteria,” Phytopathology, 81:1508-1512 (1991); Zhang, S., M. S. Reddy, N. Kokalis-Burelle, L. W. Wells, S. P. Nightengale, and J. W. Kloepper, “Lack of induced systemic resistance in peanut to late leaf spot disease by plant growth-promoting rhizobacteria and chemical elicitors,” Plant Disease, 85(8):879-884 (2001); and Murphy, J. F., G. W. Zehnder, D. J. Schuster, E. J. Sikora, J. E. Polston, and J. W. Kloepper, “Plant growth-promoting rhizobacterial mediated protection in tomato against Tomato mottle virus,” Plant Disease, 84(7):779-784 (2000). Additionally, oomycete and fungal hyphal wall fragments (see Doke, N., “Generation of superoxide anion by potato tuber protoplasts during the hypersensitive response to hyphal wall components of Phytophthora infestans and specific inhibition of the reaction by suppressors of hypersensitivity,” Physiological Plant Pathology, 23:359-367 (1983); and Anderson, A. J., “Studies on the structure and elicitor activity of fungal glucans,” Canadian Journal of Botany, 58:2343-2348 (1980)), bacterial cell wall fractions (lipopolysaccharides) (see Sequeira, L., “Mechanisms of induced resistance in plants,” Annual Review of Microbiology, 37:51-79 (1983), and phytohormones (see Cohen, Y., M. Reuveni, and A. Baider, “Local and systemic activity of BABA (DL-3-aminobutyric acid), against Plasmopara viticola in grapevines,” European Journal of Plant Pathology, 105(4):351-361 (1999); Oka, Y., Y. Cohen, and Y. Spiegel, “Local and systemic induced resistance to the root-knot nematode in tomato by DL-beta-amino-n-butyric acid,” Phytopathology, 89(12):1138-1143 (1999); and Cohen, Y. R., “Aminobutyric acid-Induced Resistance Against Plant Pathogens,” Plant Disease, 86(5):448-457 (2002)) have SIR-displayed induction capability. All publications mentioned above are incorporated herein by reference in their entireties for all purposes.
Two systemic resistance pathways have been described: 1) systemic acquired resistance, which utilizes salicylic acid as a secondary signal molecule and leads to the production of pathogenesis-related (PR) proteins (see Delaney, T. P., “Genetic Dissection of Acquired Resistance to Disease,” Plant. Physiology, 113:5-12 (1997)) and 2) induced systemic resistance, which utilizes jasmonates and ethylene as secondary signal molecules and controls disease independently of PR-protein production (see Pieterse, C. M. J., S. C. M. van Wees, J. A. van Pelt, M. Knoester, R. Laan, H. Gerrits, P. J. Weisbeek, and L. C. van Loon, “A Novel Signaling Pathway Controlling Induced Systemic Resistance in Arabidopsis,” The Plant Cell, 10:1571-1580 (1998)). All publications mentioned above are incorporated herein by reference in their entireties for all purposes.
Systemic resistance results in the activation of defenses in uninfected parts of the plant. As a result, the entire plant is more resistant to infection. The systemic resistance is long lasting and often confers broad-based resistance to different pathogens.
One of the issues surrounding systemic resistance is the occurrence of necrotic cell death at the site of application of the agent that induces systemic resistance.
Increased societal concerns related to the use of agrichemicals and genetically modified organisms as a means of managing crop diseases has prompted interest in methods of biological control. A biological control agent capable of inducing systemic resistance would provide a method of increasing disease resistance in a plant without the use of agrichemicals. Of particular interest would be a biological control agent capable of inducing systemic resistance without inducing necrotic cell death.
Thus, a need exists for new biological control agents capable of inducing systemic induced resistance in plants. A need also exists for new methods of identifying new biological control agents capable of inducing systemic resistance in plants.
Bacillus spores can potentially be used as biocontrol agents for suppressing various plant diseases. See, e.g., Emmert E A B, Handelsman J (1999) Biocontrol of plant disease-a (Gram-) positive perspective. FEMS Microbiol. Lett. 171:1-9; Shoda M (2000) Bacterial control of plant diseases. J. Biosci. Bioeng. 89:515-521; Montesinos E (2003) Development, registration and commercialization of microbial pesticides for plant protection. Int. Microbiol. 6:245-252. Spores are the preferred form for commercial delivery as spores are more efficient and less expensive to produce and more stable than freeze dried cells. Such biocontrol agents are desirable over chemical agents, which are often harmful to the environment and to humans. However, the current high costs of spore production caused by inefficiencies in culturing and fermentation methods have prevented the widespread use of Bacillus spores to control plant disease.
Many attempts have been made to enhance spore yields, particularly with Bacillus subtilis cells. See, e.g., Monteiro S (2005) A Procedure for High-Yield Spore Production by Bacillus subtilis. Biotechnol. Prog. 21:1026-1031; Hageman J H, et al., (1984) Single, chemically defined sporulation medium for Bacillus subtilis growth, sporulation, and extracellular protease production. J. Bacteriol. 160:438-441; Dingman, D W and Stahly, D P (1983) Medium Promoting Sporulation of Bacillus larvae and Metabolism of Medium Components. Appl. Environ. Microbiol. 46(4):860-869; Warriner, K. and Waites, W. M. (1999) Enhanced Sporulation in Bacillus subtilis Grown on Medium Containing Glucose:Ribose. Letters in Applied Microbiology 29:97-102; Chen, Z., et al., (2010) Greater Enhancement of Bacillus subtilis Spore Yields in Submerged Cultures by Optimization of Medium Composition Through Statistical Experimental Designs. Appl. Microbiol. Biotechnol. 85:1353-1360. Researchers have also adapted known spore culture methods in attempts to produce spores of Bacillus mycoides. See, for example, Bowen et al. (Jul. 20, 2002) The Measurement of Bacillus mycoides Spore Adhesion Using Atomic Force Microscopy, Simple Counting Methods, and a Spinning Disk Technique, Biotechnology and Bioengineering, Vol. 79(2): 170-179. However, improved methods for spore production are needed, particularly for other species within the Bacillus genus.